Malaria remains one of the most serious parasitic diseases in the third world despite the efforts to control the disease and reduce its prevalence and continued geographic spread by vector eradication and drug treatment and each year, several hundreds of millions of human beings are affected by the disease. The increasing environmental changes and the failure of classic control programmes have stimulated the search for a vaccine for the control of malaria. Naturally, one of these approaches is immunologic, and for a long time it has been hoped that immunology will provide effective vaccines for malaria. Human malaria is caused by four species of the protozoan genus, Plasmodium. The species Plasmodium falciparum is the most dangerous and malignant malaria parasite, causing acute severe infections that are often fatal, especially in young children and immigrants entering endemic areas. Thus, it is very desirable that a vaccine against P. falciparum is developed. The life cycle of P. falciparum includes different stages; in the first stage, the sporozoite stage, the parasite is brought into the blood stream by the Anopheles mosquito. The sporozoites are carried in the blood stream to the liver where they invade the hepatocytes and develop into merozoites in the course of 5-7 days. Merozoites released from infected cells start a new cycle by invading the erythrocytes. In the erythrocyte, the parasite shows an asexual multiplication which involve a maturation of the parasite through different parasite stages, the ring, the trophozoite and the schizont stage (the stage that undergoes nuclear division). When the schizont infected erythrocyte bursts, new merozoites are released. It is the disintegration of the erythrocyte which gives rise to the clinical disease.
Some merozoites, however, differentiate into gametocytes (microgametocytes and macrogametocytes), the sexual form of the parasite. Contrary to the asexual infected erythrocytes, these sexual parasite stages are able to continue the life cycle, when the infected cells, the erythrocytes, are ingested by mosquitoes during a blood meal. By fertilization in the mosquito gut, the gametocytes develop into a mobile ookinete stage. The ookinete pass through the epithel and matures into a oocyst. In the oocyst, the new sporozoites develop. These sporozoites are released and move to the salivary gland and are then ready to be injected into a new host. The parasites are haploid in most of the life cyclus as they perform a meiotic cell division shortly after fertilization.
The Anopheles mosquito is the primary vector of malaria, but the disease may also be seen after blood transfusion, i.v. injections which contaminated equipment and after transfer from an infected mother to the newborn child through the placenta.
Generally, it has proven difficult or impossible by vaccination to obtain a sufficient immunity against parasitic diseases as such due to the fact that after invasion, many parasites are capable of "cheating" the immune system of an individual by changing the appearance of the antigens or by producing substances which elicit an immune response against other components than the parasites themselves, thereby rendering the immunity obtained by the vaccination insufficient with respect to combating the development of the parasitic infection. Immunization against malaria infections has also been difficult due to the wide variety of existing different malaria parasites.
Parasites of the Plasmodium species, especially P. falciparum, are the malaria parasites which have been most intensively investigated. A number of soluble surface proteins and antigens from P. falciparum, especially in the schizont stage, have been found in sera from infected individuals (1, 3, 4, 5, 6, 7), and plasma fractions containing these antigens have been isolated and described by Jepsen and Axelsen. Typically, the antigens constitute a heterogeneous group of proteins and glycoproteins. A mixture of soluble P. falciparum antigen (antigen 1 and antigen 2) have been isolated form in vitro grown P. falciparum (2). None of the antigens 1-7 mentioned in reference 1-7 have, however, separated been isolated and purified, and the antigens have only been characterized by reference to molecular weight, glycosylation and antigenicity, their amino acid composition and possible content of epitopes as well as the nucleic acid molecules encoding the antigens have not been mentioned or indicated.
Nucleic acid sequences encoding polypeptides of various Plasmodium species have been isolated and analysed (8, 9, 10), but none of these nucleic acid sequences encode a polypeptide having a characteristic sequence GLURP and they have all been obtained following a strategy difference from the one used for isolating the DNA-sequence encoding said characteristic amino acid sequence. This will be explained in detailed in the following:
Examples of other works involving P. falciparum are described in the following patent publications:
WO 88/00597 (Kara et al.), WO 88/00595 (Epping et al.), WO 86/00620 (Koenen et al.), WO 85/03724 (Hope et al.), WO 85/00975 (Ristic et al.), WO 84/02917 (Kemp et al.), WO 84/02471 (Dubois et al.), WO 84/02472 (Dubois et al.), EP 0 252 588 Smithkline Beckman Corporation), EP 0 209 643 (Eniricerche S.p.A), EP 0 112 784 (Institut Pasteur) GB 21 99 140 (Eniricerche S.p.A.), U.S. Pat. No. 4,735,799 (Patarroyo), U.S. Pat. No. 4,707,357 (Dame et al.) and WO 85/03725 (Mach et al.), GB 2099300 (Freeman et al.), EP 0 223 665 (Vernes et al.), EP 0 136 932 (Chilbert) and EP 0 136 215 (Ristic et al.).